This invention relates to measuring total calcium in biological samples.
In biological samples, calcium ions are present either as free ions or bound to sample components such as proteins. To determine the calcium content of the sample accurately, it is necessary to measure the contributions from both forms (i.e. the "total calcium").
Total calcium has been measured colorimetrically using an acidic indicator dye, e.g., cresolphthalein complexone, whose color is sensitive to calcium ion concentration and which releases bound calcium ions. Dye and buffer solution are added to the sample separately (2 point calibration method) or combined and added together (1 point calibration method). In the latter method, the dye buffer reagent must be used within a short period of time following combination because the dye has only limited stability in the alkaline environment of the buffer solution.
Colorimetric measurement of total calcium is not compatible with the measurement of monovalent ions, e.g., sodium, potassium, or chloride because these ions cannot be measured colorimetrically.
Another way of measuring total calcium has been to use atomic absorption spectroscopy (AAS). AAS, however, is too expensive and time consuming for routine sample analysis.
Total calcium has also been measured using an ion sensitive electrode containing a calcium ionophore. In one such method, the sample is treated with a dilute acidic solution (pH=4.0 to 5.5) to release bound calcium. The calcium ionophore then binds the released ions, thereby changing the electrode's output voltage; the change is related to the calcium ion concentration. Because sodium ions cannot be measured accurately at pH values less than 6.0, this method of measuring calcium is incompatible with measurement of sodium ions. Moreover, conventional calcium ionophores are unstable in acidic conditions (pH&lt;6) because they hydrolyze and thereby lose their ability to bind calcium ions, a special chemically modified calcium ionophore that is stable under acidic conditions is used in such known methods.